Dynamic phenotypic reprogramming and chemoresistance induced by lung fibroblasts in small cell lung cancer

Small cell lung cancer (SCLC) is heterogenous in phenotype and microenvironment. Dynamic phenotypic reprogramming, leading to heterogeneity, is prevalent in SCLC, while the mechanisms remain incompletely understood. Cancer-associated fibroblasts (CAFs) possess comprehensive roles in cancer progression, while their function in phenotypic reprogramming of SCLC remain elusive. Here, we obtained transcriptome data of SCLC tissues from publicly available databases, subsequently estimated abundance of CAFs. We found CAF-abundant SCLC exhibited non-neuroendocrine (Non-NE) characteristics. Supporting this, the positive correlation of expression level of α-SMA, the CAF marker, and expression level of REST, protein typically expressed in Non-NE type SCLC, was identified in SCLC tissue arrays. Moreover, we revealed that fibroblasts inhibited NE markers expression and cell proliferation of SCLC cells in the co-culture system comprising lung fibroblasts and SCLC cells, indicating a phenotypic reprogramming from NE to Non-NE. During this process, fibroblast-derived IL-6 activated the JAK2/STAT3 signaling, upregulated c-MYC expression, and subsequently activated the NOTCH pathway, driving phenotypic reprogramming. Moreover, CAF-enriched SCLC exhibited increased immune cell infiltration, elevated expression of immune activation-related signatures, and checkpoint molecules. Our data also highlighted the chemoresistance induced by fibroblasts in SCLC cells, which was effectively reversed by JAK inhibitor. In conclusion, fibroblasts induced phenotypic reprogramming of SCLC cells from NE to Non-NE, likely contributes to inflamed immune microenvironment and chemoresistance. These findings provide novel insights into the clinical implications of CAFs in SCLC.


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Supplementary materials and methods

Cell treatments
For JAK2/STAT3 inhibitor treatment, H69 and H2227 were pre-treated with 0.8 μM AZD-1480 (MedChemExpress, NJ, USA) for 24 h, then co-cultured with fibroblasts or separately cultured with complete medium containing 0.8 μM AZD-1480, then expression of NE genes or c-MYC, HES1 or cell viability were determined.
Virus was subpackaged and stored at − 80 ° C for specified experiments.The shRNA sequence targeted to MYC was listed in Supplementary Table S4.

Cell proliferation analysis
SCLC cell line H69 and H2227 which been treated with AZD-1480 or lentivirus or not were seeded in 24-well plates at 2×10 4 cells /well and co-cultured with MRC-5 or HFL1 which were untreated or transfected with siRNA, or cultured separately.5 days later, cell viability was determined by Cell Counting Kit-8 (CCK-8) reagent (Beyotime, Beijing, China), the optical absorbance was measured at 450 nm by CLARIOstar microplate reader (BMG Labtech, Offenburg, Germany).
For clone formation test, SCLC cell line H2227 were seeded in 24-well plated at 500 cells/well and cocultured with MRC-5 and HFL1 respectively or separately cultured for 7 days.The cell clones were washed by PBS and fixed by 4% paraformaldehyde, and then stained by 0.1 % (m/v) crystal violet for 15 min.Cell clones of every well were photographed and counted by Image J software.

Cell viability assay
SCLC cells co-cultured with fibroblasts for 7 days and then seeded in 96-well plates and exposed to Cisplatin or Etoposide for another 48 h.Cell viability was measured by CCK-8 reagent.The inhibition rate was calculated with the formula as: inhibition rate=1-OD450 (drug treatment group)/OD450 (control group) ×100%.

Western blot analysis
To extract the total protein of cells, RIPA reagent supplemented with protease inhibitor and phosphatase inhibitors (Beyotime, Beijing, China) was added to SCLC cells and lysed on ice for 30 min.The concentrations of supernatant were determined using BCA protein assay kit (Beyotime, Beijing, China).The same amount of protein was separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, Darmstadt, Germany).The blots were blocked and cut prior to hybridisation with antibodies overnight at 4 ℃.The blots incubated with HRP-conjugated secondary antibodies and then chemiluminescence of blots were stimulated by ECL reagent (Beyotime, Beijing, China) and detected by GeneGnome XRQ NPC imaging system.Quantitative analysis of blots was performed using Image J software.The details of antibodies were listed in Supplementary Table S5.

RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA).ReverTra Ace qPCR RT Master Mix (TOBOYO, Osaka, Japan) was used to reverse transcribe 1 μg RNA to cDNA.Then cDNA was used to perform qRT-PCR with SYBR Green qPCR Mix (Beyotime, Beijing, China) in an Agilent Mx3000P qPCR System (Palo Alto, CA, USA).The relative mRNA expressions were calculated using the 2-∆∆Ct method.GAPDH was used as a loading control.Primers were generated by Beijing Dingguo Changsheng Biotechnology (Beijing, china) and sequences were listed in Supplementary Table S2.

Supplementary Tables
Supplementary Table S2: Sequences of primers for qRT-PCR and ChIP--8 reagent in H69 cells which separately cultured or co-cultured with fibroblasts (n=3); (c) Colony formation assay was performed to evaluate the proliferation of H2227 cells upon coculture with fibroblasts or cultured individually (n=3).(d-e) The mRNA expression of ACTA2 determined by qRT-PCR (n=3) and protein expression of α-SMA evaluated by western blot of monoand co-cultured MRC-5 cells.The blots displayed in (e) were cropped prior to hybridisation with antibodies, original images were displayed in Supplementary information.* P < 0.05, *** P < 0.001.Supplementary Figure S3Exploration of IL-6 and IL-6R in CAF-induced NE phenotypic reprogramming.(a) Secreted IL-6 from separately cultured MRC-5, HFL1, H69 and H2227 cells or co-cultured cells were determined by ELISA (n=4); (b) Expression of IL6, IGFBP1 and CCL20 was determined by qRT-PCR in MRC-5 and HFL1 cells upon transfection with indicated siRNA (n=3).(c, d) Expression of IL6 (c) and IL6R (d) was tested by qRT-PCR in MRC-5 and HFL1 cells or H69 and H2227 cells respectively upon coculture with fibroblasts or cultured individually (n=3); (e) Cell proliferation was determined by CCK-8 assay after transfection with siRNA tarted to IL6 or negative control (NC); (f) Pearson's correlation of expression of IL6 with NE score and CAF abundance in George's cohort and GSE60052 dataset.* P < 0.05, ** P < 0.01.Supplementary Figure S4 Association of CAFs with immune features in SCLC cohorts.(a, b) Pearson's correlation of CAF abundance with immune cell abundance in George's cohort (a) and GSE60052 dataset (b); (c) Expression of checkpoint molecules of high-and low-infiltration groups in GSE60052 dataset; (D-E) APM signature (d) and T-cell-inflamed GEP (e) of high-and low-infiltration groups in George's cohort and GSE60052 dataset.* P < 0.05, ** P < 0.01, *** P < 0.001.